Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Abstract

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry (IHC), is widely used in experimental studies quantifing tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected extensively by variations in the methodology used to measure vascularization including antibody selection, pretreatment with antigen retrieval and evaluation techniques. To examine this further, we examined the microvessel density and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg IHC staining. Different antigen retrieval methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, compared to F VIII RAg, anti-CD31 highlighted small vessels better. Furthermore, the microvessel density of CD31 was significantly greater than that of F VIII RAg decorated vessels (p < 0.001). The choice of antibody and antigen retrieval method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.

Antagonists of growth hormone releasing hormone (GHRH) given before whole body radiation lead to modulation of radiation response and organ-specific changes in the expression of angiogenesis

“…This method allows quantification of angiogenesis through immunohistochemical detection of CD31 [48]. In this study, the expression of angiogenesis measured by CD31 staining was consistent with the RT-PCR findings.…”

Lack of Correlation Between Microvascular Density and Pathological Features and Outcomes in Sinonasal and Oral Mucosal Melanomas

“…Again one has to consider that heterogeneous methodologies used to calculate MVD among different studies might also play a role. In our study, we chose to highlight the tumor vessels with an antibody to CD31 because it has been shown that MVD is better assessed by CD31 (or CD34) than by factor VIII-related antigen [26,27], often used in other studies. In addition, we selected random areas of the tumor [5] with the help of computer software to minimize potential inter and intraobserver variability in selecting particular areas at the growing edge of the tumor or the most vascularized ''hot spot'' areas.…”

The effects of ergot and non-ergot-derived dopamine agonists in an experimental mouse model of endometriosis

“…Four micron serial sections were deparaffinized and rehydrated through graded ethanol and rinsed in distilled water. Antigen retrieval was carried out by the autoclave method (120 8C) for 3 min in 0.5 M Tris buffer at pH 10 (for CD31; Wang et al 2008) or 0.01 M citrate buffer at pH 6.0 (for Ki67, VEGFR2 and DRD2) followed by cooling for 20 min at room temperature. Sections were gently rinsed in deionized water and then transferred to a 0.5 M Tris-based solution in 0.15 M NaCl with 0.1% v/v Triton-X-100 at pH 7.6 (TBST).…”